添加核苷对肝素黄杆菌发酵产肝素酶的影响

研究了添加核苷对肝素黄杆菌发酵产肝素酶的影响,结果发现,单种核苷的添加会抑制产酶,而复合核苷的添加则促进产酶,当4种核苷的添加比例与肝素酶mRNA中4种相应核苷酸的比例一致时,促进作用最强。通过HPLC检测,证实添加后核苷很快进入了菌体内。HPLC的结果还表明,菌体内嘧啶核苷酸和嘌呤核苷酸的合成代谢可能不平衡,这对产酶是不利的。为此,还研究了通过添加天冬氨酸以增强嘧啶核酸合成代谢的调节方式,使产酶得到了提高。     

超氧自由基与嘧啶碱基及其核苷反应的ESR研究

用自旋捕集技术和ＥＳＲ方法,以ＭＮＰ为捕集剂,研究了紫外线辐照核黄素产生超氧自由基等活性氧与嘧啶碱基及其核苷的反应,确定了尿嘧啶,胞嘧啶和胸腺嘧啶及其核苷所产生的自旋加合物自由基的类别,讨论了自由基的形成机制,揭示了超氧自由基与嘧啶碱基及其核苷的反应是不是直接进行的,而是通过羟基自由基来实现的。     

嘧啶核苷磷酸化酶与抗癌药物治疗效果的评价

嘧啶核苷磷酸化酶(PyNPase)是嘧啶核苷补救代谢途径中的关键酶,广泛分布于微生物及动物组织细胞中。近几年来,很多学者对PyNPase在抗癌药物合成和癌症治疗方面的作用及其临床应用进行了广泛的研究。本文综述了PyNPase与肿瘤患者临床病理特征、抗癌药物评价等之间的关系。     

四氢嘧啶微生物合成与应用研究进展

极端环境微生物定义了生命的边界。为了适应各种极端环境,极端环境微生物通过合成许多独特的活性化合物来保护自己。四氢嘧啶就是其中一种代表性的保护性物质。它最早是从极端嗜盐菌中发现的,作为调节细胞渗透压的一类相容性溶质,可以帮助微生物适应高盐等恶劣环境。研究发现四氢嘧啶不仅是一种重要的渗透压调节剂,还是一种高效的生物保护剂,可以帮助蛋白、核酸、生物膜乃至整个细胞对抗高温、干燥、冷冻和辐射等多种逆境。因此,四氢嘧啶在生物保护、生物医药和生物科技等众多领域展现出广阔的商业化应用前景。随着合成生物学和代谢工程技术的快速发展,传统的嗜盐菌四氢嘧啶生产方法已逐步被产率更高、环境更友好的生物工程菌及技术所取代。本文围绕四氢嘧啶的微生物合成及其应用研究进行综述,为后续四氢嘧啶的开发和应用提供重要参考。     

高聚物高效液相色谱柱分离分析嘧啶和核苷

建立了使用高聚物型色谱柱,反相高效液相色谱法(RP-HPLC)测定5种嘧啶和核苷的方法,样品经甲醇溶解,以p H为4.4的0.01 mol/L KH2PO4缓冲盐溶液和甲醇为流动相进行梯度洗脱,流速1.0 m L/min,温度20℃,紫外检测波长251 nm。考察了流动相中不同因素对分离分析的影响,实现了5种嘧啶和核苷混合物的有效分离。柱效可达37 400 N/m,优于常规的C18-硅胶色谱柱(柱效为21 400 N/m),为该高聚物填料在嘧啶和核苷化合物检测方面的广泛应用提供了有关依据。     

相容性溶质四氢嘧啶及其羟基化衍生物的研究进展

四氢嘧啶类化合物是嗜盐以及耐盐菌胞内合成的一类能够抵御外界高盐胁迫的相容性溶质,概述了四氢嘧啶及其衍生物的理化特征以及在嗜盐微生物中抵御外界高渗透压的作用机理,主要阐述了四氢嘧啶类相容性溶质的生物合成途径、膜运输机理、分泌释放机制、高密度发酵生产等方面在细胞、分子水平上的最新研究进展以及前景展望。并且综述了四氢嘧啶类在精细化工、生物医药及生物制造等行业的应用研究以及发展前景,探讨了未来的研究方向。     

渗透压冲击下中度嗜盐菌Halomonas sp. Y四氢嘧啶类相容性溶质的合成与释放

【背景】四氢嘧啶类物质在高温、冷冻和干燥等逆境条件下,对酶、蛋白质、核酸及整个细胞具有良好的保护作用,已经应用于酶制剂、生物医药及护肤品等相关领域。目前此类物质只能依赖中度嗜盐菌采用细菌泌乳工艺进行商业化生产,因此四氢嘧啶类高产菌株及其发酵技术的研究日益受到国内外研究者关注。【目的】分离获得高产合成四氢嘧啶类相容性溶质的中度嗜盐细菌,研究渗透压冲击对其胞内四氢嘧啶合成与释放的影响,探索细菌泌乳法制备四氢嘧啶的可行性。【方法】采用涂布平板法分离中度嗜盐菌,对分离菌株进行形态、生理生化和16S rRNA基因序列分析,鉴定其种属;采用高效液相色谱法(HPLC)和质谱法(MS)分析四氢嘧啶类物质,细菌泌乳法制备四氢嘧啶类物质。【结果】从盐池土样中分离到一株以四氢嘧啶类物质为主要相容性溶质的中度嗜盐菌Y,鉴定为盐单胞菌(Halomonas sp.)Y。盐单胞菌Y能在NaCl质量浓度为10-250 g/L的培养基中生长,最适生长的NaCl浓度为100 g/L;HPLC-MS测试结果证明盐单胞菌Y可同时合成四氢嘧啶和羟基四氢嘧啶2种相容性溶质,在最适生长的盐浓度下其合成量分别达175.5 mg/g和47.9 mg/g;在NaCl质量浓度为0-30 g/L的低渗溶液中胞内四氢嘧啶类物质经5 min即可达到最大释放率,而细菌泌乳工艺中最适合诱导四氢嘧啶释放的低渗溶液为质量浓度为10 g/L的NaCl溶液;采用细菌泌乳工艺制备四氢嘧啶,经连续11轮的高渗/低渗冲击,四氢嘧啶总合成量为6.0 g/L,总释放量为5.7 g/L,平均释放率为64.5%,底物转化率为128.9 mg/g。【结论】盐单胞菌Y是一株较高产合成四氢嘧啶类的中度嗜盐菌,能够耐受反复的渗透压冲击,采用细菌泌乳工艺显著提高了四氢嘧啶的制备效率。     

乙酰短杆菌中核苷磷酸化酶的性质研究

目的：以乙酰短杆菌完整细胞为酶源,研究不同条件下核苷磷酸化酶的性质。方法：将乙酰短杆菌湿茵体置于不同保藏温度及在不同种类缓冲溶液中考察其稳定性;在有或无核保护下核苷磷酸化酶对热的稳定性;并设计核苷的磷酸解反应或合成反应,测定核苷磷酸化酶的活力及酶促反应的袁观米氏常数。结果：乙酰短杆菌中的核苷磷酸化酶经低温保藏可以保持较长时间的稳定性;茵体在60℃处理1小时即失去核苷磷酸化酶的活力,但是添加胸腺嘧啶有明显的保护作用;茵体中核苷磷酸化酶的合成能力明显大于磷酸解能力;对尿苷和5-甲基尿苷的表观米氏常数和最大反应速率分别为16．7、11．4mm01／L,0．0063、0．0041mmol／L．min。结论：含核苷磷酸化酶的乙酰短杆菌完整细胞作为酶源,在低温下可以长时间保藏,反应中的碱基对核苷磷酸化酶的抗热性有益,该菌种可以作为工业上核苷磷酸化酶的来源。     

枯草芽孢杆菌cdd基因敲除及对胞苷发酵的影响

目的:通过敲除出发菌株上的胞苷脱氨酶基因,阻断嘧啶代谢通量由胞苷流向尿苷和尿嘧啶,选育胞苷产生菌。方法:采用同源重组的方法敲除枯草芽孢杆菌TS8的胞苷脱氨酶基因cdd,并通过遗传稳定性实验验证其缺失标记和胞苷产量,通过摇瓶发酵实验对比出发菌株和缺失株的产苷水平。结果:cdd基因缺失菌株TSb发酵72h,发酵液中胞苷产量达到1.72g/L,与原始菌株相比提高了44.19%,且遗传性状稳定。结论:cdd基因的缺失可有效阻断嘧啶代谢通量由胞苷流向尿苷和尿嘧啶,提高胞苷产量。     

中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析

中度嗜盐菌Bacillus alcalophilus DTY1分离自晋西北黄土高原盐碱土壤, 能够产生耐盐相关的相容性溶质四氢嘧啶。为了研究四氢嘧啶的功能, 克隆了DTY1菌株四氢嘧啶合成基因簇ectABC。ectA、ectB和ectC分别编码169、428和132个氨基酸的肽链, 分别与B. halodurans C-125中的二氨基丁酸乙酰基转移酶(EctA)、二氨基丁酸氨基转移酶(EctB)、四氢嘧啶合成酶(EctC)同源性达59%、81%和81%。将携带该基因簇的4.0 kb片段转入蜡质芽孢杆菌B. cereus Z后, 芽孢杆菌的耐盐度显著提高。HPLC检测发现, 在1.0% NaCl浓度下, 转化菌B. cereus Z-E菌株生成70.1 mg/g四氢嘧啶, 而在5.0%的NaCl浓度下四氢嘧啶的产量高达118.6 mg/g, 显著高于B. alcalophilus DTY1的四氢嘧啶产量。而且随着盐浓度的提高, 四氢嘧啶的合成量也随之提高。由此证明四氢嘧啶参与中度嗜盐菌重要的渗透调节, ectABC的表达受盐诱导。     

Mechanism of uridine production by Bacillus subtilis mutants

Summary Pyrimidine analogue-resistant mutants of Bacillus subtilis were found to produce a large amount of uridine. One of them accumulated 55 mg/ml of uridine in culture medium. The changes in enzymes involved in the metabolism of uridine 5-monophosphate (UMP) were examined with this mutant. All six enzymes of de novo UMP biosynthesis were completely free from regulation by uridine compounds, and the activities of these enzymes were 16- to 30-fold higher than those of the enzymes of the parental strain. In the mutant strain, the level of uridine phosphorylase, responsible for converting uridine to uracil, was extremely low, compared with that of the parental strain. No apparent change was observed between the strains in the activity of UMP dephosphorylation or uracil phosphoribosyltransferase. The implication of these findings is discussed in relation to the overproduction of uridine by the mutant.Microbial production of uridine. Part III     

Genome shuffling在酿酒酵母菌种选育中的应用

Genome shuffling(基因组改组)作为一种新型的菌种选育方法,与常见的育种方法相比,具有快速有效、简单易行和实用性强等特点,近年来不断应用于各种工业微生物菌种的改良研究中。论述了Genome shuf-fling的产生与原理、技术过程及其在酿酒酵母中的应用和发展前景。     

Simultaneous Protection of 3′- and 5′-Hydroxyls of Ribonucleosides with Di-t-Butoxydichlorosilane

Abstract

Di-t-butoxydichlorosilane was found to protect simultaneously 3′- and 5′-hydroxyls of uridine. The preliminary results on the dialkoxysilanediyl group introduction, properties and applications are presented.     

Comparative effects of 5-fluorouracil on strains of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Comparative effects of 5-fluorouracil on strains of Bacillus megaterium. J. Bacteriol. 87:1011-1018. 1964.-Growth of Bacillus megaterium strain KM is severely inhibited by 5-fluorouracil (FU). Both thymidine and uridine are required to overcome this inhibition. The addition of uridine alone to a FU-inhibited culture permits good ribonucleic acid (RNA) and protein synthesis for the first 2 hr, but rather poor deoxyribonucleic acid synthesis. Uridine enhances the bactericidal effect of FU, promoting a decrease in the viable count of from 4 to 5 decades in 5 hr. Death begins after a 1-hr lag and is accompanied by hydrolysis of RNA and cell lysis, commencing during the 2- to 5-hr interval. The combination of FU and uridine is not bactericidal, when a methionine auxotroph is deprived of its required amino acid. Substrains of KM, partially resistant to FU, were isolated. Strain T(2) requires only thymidine to overcome the inhibitory effects of FU, whereas strain FU/2 requires only uridine. With a uridine auxotroph of strain KM, FU partially replaces uridine by permitting a small, but reproducible, increase in the amount of protein.     

后基因组时代基因组重排在微生物菌种选育中的应用及展望

基因组重排作为一种实用高效的育种技术,在缺乏遗传背景认知和可操作遗传体系等条件下,可以突破微生物种属间的限制,经过多轮递推的原生质体融合来加速其人工定向进化,在微生物菌种改良及代谢产物开发和产业化等研究领域得到了广泛应用。步入后基因组时代,快速发展的组学和生物信息学使基因组重排成为连接各种微生物育种方法的重要纽带,为我们深入探索微生物复杂的代谢网络和全局调控机制,更为精准地实施对微生物的人工调控和定向进化提供了契机。本文系统性地回顾了近年来基因组重排在微生物菌种选育中的应用研究,尤其针对围绕其开展的组学研究进行了详细阐述,并对基因组重排与组学、生物信息学和合成生物学等新兴技术的联合应用进行了展望。     

Proteolytically induced changes in the molecular form of the carbamyl phosphate synthetase-uracil-aspartate transcarbamylase complex coded for by the URA2 locus in Saccharomyces cerevisiae.

When a uracil-auxotrophic yeast strain is grown under uracil-limiting conditions, the aspartate transcarbamylase activity found in crude extracts shows a variation in sensitivity to feedback inhibition by uridine 5'-triphosphate. In this study we correlated this variation with changes in the molecular form of the carbamyl phosphate synthetase-uracil-aspartate transcarbamylase complex. Carbamyl phosphate synthetase-uracil (molecular weight, 240,000) and uridine 5'-triphosphate-insensitive aspartate transcarbamylase (molecular weight, 140,000) were present separately in extracts from cells collected in the early exponential phase; this was in contrast to the presence of a single high-molecular-weight form (molecular weight, about 900,000) bearing both activities in extracts from stationary-phase cells. The lack of sensitivity to uridine 5'-triphosphate by aspartate transcarbamylase was delayed by adding uridine 5'-triphosphate before cell disruption and was prevented completely by adding phenylmethylsulfonyl fluoride. Thus, this event was attributed to a transient serine protease activity detected only in early exponential-phase cell extracts. However, even in the presence of phenylmethylsulfonyl fluoride, a sucrose density gradient analysis in the absence of uridine 5'-triphosphate revealed a change in the aggregation state of the complex which might have occurred in vivo. None of these events was observed in extracts from cells that lacked protease B activity (strain HP232-2B).     

Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.     

Errata

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterized the transport mechanism of uracil and uridine. Mutant U^?, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR^?, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U^?UR^?isolated from a culture of mutant U^?, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR^?R^? was isolated from a culture of mutant UR^?, and is resistant to high concentration of showdomycin. This mutant is defective for transport of intact uridine and in addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U^? and UR^?, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these process.     

5-Fluoropyrimidine-resistant mutants of pneumococcus

Three classes of 5-fluorpyrimidine-resistant mutants of Diplococcus pneumoniae have been characterized. The mutant strain upp is resistant to high concentrations of the fluoropyrimidine bases fluorouracil (FU) and fluorocytosine (FC); strain upp has a defective uridine monophosphate pyrophosphorylase. The mutant strain udk is resistant to inhibition by fluorouridine (FUR) and exhibits defective uridine kinase activity. The mutant strain fun is resistant to inhibition by the nucleosides fluorodeoxyuridine, fluorodeoxycytidine, and FUR, but shows normal activity for all pyrimidine pathway enzymes tested. This strain may be defective in the activity of a transport system that governs the cellular uptake of pyrimidine ribo- and deoxyribonucleosides. Biochemical studies on wild-type and fluoropyrimidine-resistant pneumococci are discussed with respect to the transport and early metabolism of preformed pyrimidine precursors by this organism.     

Two nucleoside uptake systems in Lactococcus lactis: competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of (33)P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H(3)BO(3)-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K(m) for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.